Efficient artificial breeding and intermediate cultivation method for holothuria leucospilota

Abstract

The invention discloses an efficient artificial breeding and intermediate cultivation method for holothuria leucospilota. The holothuria leucospilota with mature gonad is firstly dried in the shade and then transferred into seawater, sodium sulfite is added to make water oxygen deficit, and spawning induction is stimulated. According to the physiological habits of the holothuria leucospilota, hypoxia suffocation stimulation is carried out on the holothuria leucospilota by using sodium sulfite, and a very good spawning induction effect is achieved. Compared with an existing traditional tropical sea cucumber artificial spawning induction method, such as a running water stimulation method and a shade drying method, with the highest spawning induction rate of 13.3-18.0%, the effect of sea cucumber spawning induction is more obvious (the spawning induction rate reaches up to 75-81%) by means of the method. The sodium sulfite has no obvious toxicity to the parent sea cucumber and is low in price; a traditional spawning induction method must be operated at night, but the method has no requirement for spawning induction time, has the advantages of being high in operability, easy to popularize and capable of reducing parent parameter quantity and providing stable fertilized egg supply for large-scale sea cucumber breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and more specifically, relates to a method for efficient artificial breeding and intermediate cultivation of sea cucumbers. Background Art

[0002] Sea cucumbers belong to echinoderms and are widely distributed in the intertidal zones of the world's oceans to the depths of 10,000 meters. Except for a few floating and planktonic flat-footed orders, most of them live benthic, mainly distributed in reefs, mud and seaweed-covered areas. According to statistics, there are more than 1,400 species of sea cucumbers in the world, of which more than 40 are edible. Sea cucumbers are a traditional tonic food in my country and one of the eight delicacies of seafood. They have huge market demand in Asia. At present, the sea cucumbers with higher economic value and larger breeding scale in my country are the sea cucumbers of the north (Apostichopus japonicus). Due to the lack of artificial breeding technology, the breeding efficiency of economic sea cucumbers in tropical waters is low. They have always relied on catching wild groups to meet market demand.

[0003] Sea cucumber fishery has a history of more than 1,000 years. With the improvement of human consumption level, the global sea cucumber catch has increased rapidly in the past few decades. The world sea cucumber trade has developed into a huge industry worth billions of dollars across more than 70 countries. However, under the pressure of overfishing, environmental pollution and climate change, the resources of sea cucumbers in natural waters around the world have shown a rapid decline in recent years, and sea cucumber populations in some seas are even on the verge of extinction. In recent years, with the overexploitation of high-value sea cucumber species, some low-value sea cucumber species, such as the jade foot sea cucumber (Holothurialeucospilota), have been listed as important fishing targets. Due to long-term unreasonable fishing, the jade foot sea cucumber resources in natural waters at home and abroad have been severely damaged. Therefore, artificial breeding and proliferation and release are imminent.

[0004] Sea cucumbers are widely distributed in tropical and subtropical waters of the central and western Pacific, Asia, and the Indian Ocean. In my country, they are mainly distributed in the coastal waters of the southern East China Sea and the South China Sea. The body wall, gonads, and intestines of sea cucumbers are edible, and they are rich in a variety of bioactive substances, such as saponins, collagen, and polysaccharides, which have anti-tumor, anti-thrombotic, anti-aging, and immunity-enhancing effects. In natural waters, sea cucumbers play an important role in the habitat environment. They can transport a large amount of sediment through feeding and assimilation, accelerate the mineralization of organic matter in surface sediments, improve the circulation efficiency of nutrients in the seabed ecosystem, and effectively improve the benthic environment. In addition, sea cucumbers can also eat the leftover bait and feces of farmed animals. Mixing them in fish and shrimp farming systems can help improve the farming system environment and increase the growth rate and survival rate of farmed objects.

[0005] At present, one of the important ways to restore sea cucumber resources at home and abroad is to increase the number of stocks, which requires a stable supply of seedlings. Compared with economic species such as Apostichopus imitus and Holothuria scabra, the artificial breeding technology of sea cucumber is not yet mature. There are still problems such as poor induced spawning effect, cumbersome operation, high cost, long planktonic larvae cultivation cycle, frequent enemies, and low metamorphosis attachment rate. In addition, there is no report on the intermediate cultivation of sea cucumber seedlings. In order to promote the industrialization of sea cucumber farming, it is necessary to optimize its artificial breeding technology.

[0006] We have found in the long-term practice that the gonads of the sea cucumbers in natural waters begin to mature in July and August, and their breeding is greatly affected by time and weather. In production, if the time of harvesting the sea cucumbers is too early, the gonads are immature, and if it is too late, the sea cucumbers have already been released, and they cannot be used for artificial breeding. In addition, when the gonads of the sea cucumbers mature, if there is a storm, they will be released synchronously. Therefore, if the time of harvesting the sea cucumbers is not well grasped, it will not be possible to provide a stable supply of fertilized eggs for artificial breeding. In the process of inducing sea cucumbers to ovulate, we found that before cloudy days and storms, the air pressure decreases and the dissolved oxygen concentration in the seawater decreases. At this time, the success rate of inducing sea cucumbers to ovulate is higher. Therefore, the low oxygen environment is an important factor in inducing sea cucumbers to ovulate; in addition, when the male sea cucumbers in the induced ovulation system release sperm, the female sea cucumbers will release eggs. Therefore, sperm is also an important factor in inducing sea cucumbers to ovulate. In production practice, we also found that the juvenile sea cucumbers are quite different from other species such as the imitation sea cucumbers. They are more sensitive to the environment during the attachment metamorphosis stage. The changes in water quality and water flow caused by the placement of corrugated board attachment substrates at this stage will cause the juveniles to sink to the bottom quickly and die in large numbers. However, delaying the placement of attachment substrates and allowing most of them to settle naturally will increase the efficiency of metamorphosis and attachment. In the intermediate growth stage, if only foods with high organic matter content such as yeast and algae powder are added to the bait, the juvenile sea cucumbers will have a low feeding rate and grow very slowly. However, adding ingredients with low nutrient content such as sea mud will greatly increase the feeding rate and growth efficiency of the juvenile sea cucumbers. Summary of the invention

[0007] The purpose of the present invention is to provide a method for efficient artificial breeding and intermediate cultivation of sea cucumbers. The method optimizes each key step of the sea cucumber seedling cultivation process, including the key links of parent ginseng ripening and induced spawning, water quality control, enemy and pest control, planktonic larvae cultivation and attachment, and juvenile ginseng intermediate cultivation, greatly improving the efficiency of artificial breeding and intermediate cultivation, and reducing production costs. The method can effectively regulate the artificial breeding time of sea cucumbers and even carry out off-season seedling cultivation, overcome the unstable induced spawning effect, cumbersome operation, damage to parents, high cost, and a series of problems such as difficult to regulate seedling water quality, frequent enemy and pests, low juvenile metamorphosis rate and attachment rate, and slow growth of juvenile ginseng, and realize the efficient breeding and intermediate cultivation of sea cucumber seedlings, which has a very broad application space in the field of artificial breeding and aquaculture of tropical sea cucumbers.

[0008] The first object of the present invention is to provide a method for inducing labor of sea cucumber, which comprises the following steps:

[0009] The sea cucumbers with mature gonads are first dried in the shade and then transferred to seawater. Sodium sulfite is added to make the water hypoxic to stimulate induced labor.

[0010] Preferably, after adding sodium sulfite to cause water hypoxia and stimulate induced labor, the seawater is replaced and sperm of sea cucumber is added for induction.

[0011] Preferably, the drying in the shade takes 20-30 minutes, more preferably 30 minutes.

[0012] Preferably, 0.1 g / L sodium sulfite is added to cause anoxic conditions in the water, stimulating the production for 20-30 minutes, more preferably 30 minutes.

[0013] The second object of the present invention is to provide a method for efficient artificial breeding and intermediate cultivation, including water quality control, ripening and spawning of parent ginseng, prevention and control of harmful organisms, cultivation and attachment of planktonic larvae, and intermediate cultivation of juvenile ginseng;

[0014] The described induced labor by ginseng is the induced labor method of the above-mentioned sea cucumber.

[0015] Preferably, the method specifically comprises the following steps:

[0016] A. Water quality control: The natural seawater used must meet the second-class seawater quality standard and must be filtered and sterilized by ultraviolet light before use;

[0017] B. Ripening and induced spawning of sea cucumbers: Wild sea cucumbers are collected in advance before the breeding season as a breeding group, and the parent sea cucumbers are temporarily kept in a ripening pool in a greenhouse. The water temperature in the ripening pool is higher than that in the natural sea area. The sea cucumbers feed on benthic diatoms in the pool. At the same time, they are fed with sea mud and Sargassum powder as supplementary bait to promote the maturation of the sea cucumber gonads. The method of induced spawning is to place the parent sea cucumber in a basket to dry in the shade for 30 minutes, and then transfer it to a plastic bucket filled with filtered seawater. Sodium sulfite is added to make the water body hypoxic. The stimulation lasts for 20-30 minutes, and then the seawater is replaced. The sperm obtained by dissection is used for induction. After 2-4 hours, the parent sea cucumbers lay eggs, and the parent sea cucumbers are removed after they stop ovulating. The fertilized eggs are filtered through a 100-mesh silk screen to remove large impurities, and then concentrated with a 400-mesh silk screen to remove most of the excess sperm. The density of the fertilized eggs is adjusted to 0.5-0.7 / mL for hatching and larval cultivation;

[0018] C. Cultivation of juvenile sea cucumbers: The juvenile sea cucumbers are cultivated in a closed environment indoors. During the entire cultivation process, moderate aeration is maintained to keep the juveniles in a suspended state. They are fed with a certain ratio of Chaetoceros liquid and baker's yeast suspension. According to the turbidity of the aquaculture water, 1 / 2 of the water is replaced by siphoning every 5-7 days. Dead juveniles, residual bait and feces at the bottom of the bucket are regularly removed by siphoning.

[0019] D. Sea cucumber larvae attachment and intermediate growth: When most of the larvae develop into barrel-shaped larvae, reduce the amount of inflation appropriately, and continue to culture until the number of planktonic larvae in the water body decreases significantly, then start to release polyethylene corrugated board attachment base, with a density of 2-3 frames / m 2 ;

[0020] After the sea cucumber larvae attach, the feed is changed to benthic diatoms, marine red yeast, Chlorella powder, and Sargassum powder. Marine red yeast, Chlorella powder, and Sargassum powder are mixed in a mass ratio of 1:1:2 and fed once a day. The feed amount increases from 1ppm to 5ppm as the seedlings grow. Benthic diatoms are fed once every 3 days, and the feed amount also increases with the growth of the seedlings. After 3 hours of feeding, long-flowing water is used to keep the water quality stable. Two weeks after attachment, when the body length of the juvenile sea cucumbers reaches about 1mm, an appropriate amount of fresh sea mud is added daily. During the intermediate cultivation period, the number of daily feedings can be increased to increase the growth rate of the sea cucumbers, and large-sized sea cucumbers can be continuously cultivated.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses a ripening pool in a greenhouse to culture and ripen sea cucumbers. The greenhouse ceiling is light-transmitting, which can effectively increase the temperature of the ripening pool. In addition, artificial feeding is beneficial to the early maturation of the sea cucumber gonads. The indoor environment is relatively stable and easier to retain the sea cucumber gonads, effectively avoiding the natural ovulation of sea cucumbers caused by typhoons and rainstorms in natural sea areas. Some sea cucumbers that have been induced to ovulate can be induced to ovulate multiple times after the culture pond has recovered for a period of time.

[0023] (2) The present invention uses sodium sulfite to stimulate the sea cucumber to induce hypoxia and asphyxiation according to its physiological habits, and obtains a very good induction effect. Compared with the existing traditional tropical sea cucumber artificial induction methods such as running water stimulation method and shade drying method, the

[0024] Compared with the induction rate of 13.3-18.0%, the induction effect of sea cucumbers using this method is more obvious (the induction rate is as high as 75-81%).

[0025] Sodium sulfite has no obvious toxicity to the parent sea cucumber and is cheap; the traditional method of inducing labor must be performed at night, while the present invention has no requirements for the time of inducing labor, has strong operability, is easy to promote, reduces the amount of parent sea cucumbers, and can provide a stable supply of fertilized eggs for large-scale sea cucumber breeding.

[0026] (3) The present invention delays the time of placing the attachment substrate to reduce the interference of water flow changes on the larvae. In traditional sea cucumber seedling cultivation, the attachment substrate is placed when the proportion of barrel-shaped larvae reaches 30-50%. According to the ecological habits of the larvae of the sea cucumber, the present invention waits until some larvae naturally attach to the bottom of the barrel and their ability to adapt to the environment is enhanced before placing the attachment substrate. This can effectively avoid the large-scale death of larvae in the attachment metamorphosis period caused by sudden changes in the environment due to the addition of the attachment substrate.

[0027] (4) The present invention uses a 100-mesh gauze to cultivate benthic diatoms. Compared with traditional polyvinyl chloride corrugated plates, the gauze is cheap, easy to operate, and has a large specific surface area, which is conducive to accelerating the attachment and growth of benthic diatoms. The attached benthic diatoms can be easily peeled off, effectively supplementing fresh bait for juvenile sea cucumbers.

[0028] (5) The present invention uses marine red yeast, chlorella powder, sargassum powder and sea mud as bait for young sea cucumbers. There are few related studies on the bait for young sea cucumbers. Traditional sea cucumbers use algae powder, benthic diatoms, yeast and probiotics as bait during the intermediate cultivation period. The addition of sea mud in the present invention is more in line with the feeding habits of young sea cucumbers and can significantly accelerate the growth of young sea cucumbers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The sea cucumbers are ripened in the ripening pool in the greenhouse, and the ceiling of the workshop is transparent;

[0030] Figure 2 The gonads of mature sea cucumbers, the left is female and the right is male, photographed on August 1, 2023;

[0031] Figure 3 For indoor temporary breeding pools, the ceiling of the workshop is not transparent;

[0032] Figure 4These are the gonads of parent ginseng in the temporary holding pond. The gonads can be restored multiple times, and some parents can be kept for a long time. The left picture was taken on December 8, 2023, and the right picture was taken on March 15, 2024;

[0033] Figure 5 To optimize the induced spawning process; A. Shade drying for 30 minutes, B. Sodium sulfite hypoxia stimulation for 20-30 minutes, C. Hypoxia asphyxiation reaction of sea cucumbers, D. Sperm dissection to induce spawning;

[0034] Figure 6 To collect the fertilized eggs, first use a 100-mesh screen to filter out large particles of impurities, and then use a 400-mesh screen to collect the eggs and remove excess sperm;

[0035] Figure 7 The development status of the planktonic larvae of sea cucumbers changes with time, 2d represents the second day after fertilization, and so on;

[0036] Figure 8 The changes of body length of planktonic larvae of sea cucumber with time;

[0037] Fig. 9 The left picture shows the diatom culture tank, and the right picture shows the benthic diatoms on the gauze are rubbed into clean seawater as bait for juvenile sea cucumbers.

[0038] Fig.10 After the corrugated board substrate was added, the larvae were swept to the bottom of the water by the current, causing mass death;

[0039] Fig.11 For the intermediate cultivation of juvenile sea cucumbers, benthic diatoms, sea mud, algae powder and marine red yeast are added during the period;

[0040] Fig.12 This is a young ginseng after being attached for 6 months. The picture was taken on April 11, 2024.

[0041] Fig.13 In order to compare the changes of juvenile sea cucumber body length over time with and without sea mud, in addition to sea mud, benthic diatoms, algae powder and yeast were added to both groups.

[0042] Fig.14 The left picture shows the sea mud added during the growth process of young sea cucumbers, and the right picture shows the sea sand added;

[0043] Fig.15 This is the experimental site for using sea mud alone, sea sand alone, sea mud + Sargassum powder, and sea sand + Sargassum powder as bait for juvenile sea cucumbers. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the methods and devices used in the present invention are commonly used methods and devices in the art.

[0045] Example 1 Ripening and induced spawning of sea cucumber parents

[0046] Starting from July 2023, artificial breeding experiments of Yuzu sea cucumbers were carried out at the Shenzhen Experimental Base to further optimize the ripening and induction conditions of Yuzu sea cucumbers. The relevant process is as follows:

[0047] Ginseng ripening: Ginseng was caught from Judiaosha, Daya Bay on July 11 and temporarily kept in an indoor ripening pond ( Figure 1 ), the sea cucumber's gonads are not yet mature. During the temporary rearing period, the sea cucumber feeds on benthic diatoms that grow naturally at the bottom of the pond. A mixture of sea mud and Sargassum powder is fed weekly to enhance nutrition. The water quality is kept stable by using long-flowing water. The water temperature in the pond (28-30℃) is slightly higher than that in the natural sea (27-29℃). In early August, the sea cucumber's gonads were fully mature ( Figure 2 ), and conducted four induced labor experiments on August 7-8 and August 18-19, 2023 (Table 1).

[0048] 1. Shade-drying to induce spawning: Place the artificially ripened ginseng in a basket and shade-dry for 30 minutes ( Figure 5 -A), and then transferred to a plastic bucket filled with filtered seawater;

[0049] 2. Sodium sulfite hypoxia stimulation and induction of labor: transfer the ginseng to a plastic bucket filled with filtered seawater, add ~0.1g / L sodium sulfite to make the water hypoxic, and continue to stimulate for 20-30 minutes ( Figure 5 -B), and then replace with clean seawater;

[0050] 3. Shade drying + sodium sulfite hypoxia stimulation and induction of labor: Place the ginseng in a basket and shade dry for 30 minutes ( Figure 5 -A), and then transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to make the water hypoxic, and the stimulation continued for 20-30min ( Figure 5 -B), and then replace with clean seawater;

[0051] After the operation of Example 1 of the present invention, it was found that the gonads of the parent sea cucumbers were all mature. By comparing shade drying alone, sodium sulfite anoxic stimulation and the combined stimulation of the two, it was found that shade drying + sodium sulfite anoxic stimulation had a better induction effect. A total of 4 induction experiments were carried out, with a success rate of 75-81%, and about 20 million fertilized eggs were obtained. The average egg laying amount of female ginseng was about 2 million per head; the induction rate using this method was much higher than the induction rate of shade drying alone (0) and the induction rate of sodium sulfite anoxic stimulation alone (43%).

[0052] The induced spawning rate (%), the number of fertilized eggs (pieces) and the average egg production (pieces / head) were calculated as follows:

[0053] Oxygenation rate (%) = (ejaculation sea parameter amount + ovulation sea parameter amount) / oxytocin sea parameter amount * 100

[0054] Number of fertilized eggs = number of fertilized eggs in 6-well plate / 5*(100 or 200 dilution factor)*volume of fertilized egg water

[0055] Average egg production (pieces / head) = number of fertilized eggs / ovulation sea parameter

[0056] Note: The method for counting the number of fertilized eggs is to take out the parent sea cucumber after it stops ovulating, first use a 100-mesh sieve to filter out large particles of impurities, and then use a 400-mesh sieve to collect the fertilized eggs ( Figure 6 ), pour the fertilized eggs into clean seawater, use a measuring cup to count the water volume (mL), gently stir to mix, use a pipette to transfer 5mL of liquid to a beaker filled with 500mL or 1000mL of clean seawater, stir evenly and then pipette 5mL of liquid several times into a 6-well plate to count the fertilized eggs.

[0057] Embodiment 2 Yuzu sea cucumber parent induced spawning

[0058] Sea cucumber collection: On September 11, the sea cucumber was caught by diving in the Judiaosha area of ​​Daya Bay (Daya Bay Catch 1). A typhoon had just occurred a few days before the catch. Referring to the method in Example 1, the sea cucumber was dried in the shade for 30 minutes and stimulated with sodium sulfite anoxic for 30 minutes to induce spawning. However, no spermatogenesis or ovulation occurred. Dissection revealed that the gonads of the sea cucumber had all degenerated (Table 1).

[0059] Example 3 Ripening and induced spawning of sea cucumber parents

[0060] Parent sea cucumber collection: On September 17, the Yuzu sea cucumber was caught by diving from the Dapeng Bay waters of Shenzhen (Dapeng Bay catch 2). The sea area was less affected by the typhoon. At this time, about 50% of the sea cucumbers had full gonads. The sea cucumbers were temporarily kept in an indoor pool, which was normally aerated during the period. The sea cucumbers mainly fed on benthic diatoms and sediments that grew naturally at the bottom of the pool. The sea cucumbers were induced to spawn from September 21 to 23, 2023 (Table 1);

[0061] Shade drying + sodium sulfite hypoxia stimulation and induction of labor: Place the sea cucumber in a basket and shade dry for 30 minutes, then transfer it to a plastic bucket filled with filtered seawater, add sodium sulfite to make the water hypoxic, continue stimulation for 30 minutes, and then replace with clean seawater;

[0062] Through the operation of Example 3 of the present invention, it was found that shade drying + sodium sulfite hypoxia stimulation had a better effect on inducing labor of naturally mature sea cucumber parents in the wild. A total of 3 induction experiments were carried out, with a success rate of 2 times, and about 4.7 million fertilized eggs were obtained.

[0063] Embodiment 4 Yuzu Sea Cucumber Parent Induced Labor

[0064] Parent sea cucumber collection: On September 22, the sea cucumbers were caught by diving in the Dapeng Bay waters of Shenzhen (Dapeng Bay catch 3). At this time, the sea cucumbers had full gonads, and they were temporarily kept in indoor pools, during which they were normally aerated. The sea cucumbers mainly fed on benthic diatoms and sediments that grew naturally at the bottom of the pool. They were induced to spawn on September 24-25, 2023, and were induced to spawn again after being temporarily kept for a period of time after induction (Table 1);

[0065] Shade drying + sodium sulfite hypoxia stimulation + sperm induction and induced ovulation: Place the sea cucumber in a basket and shade dry for 30 minutes, add ~0.1g / L sodium sulfite to make the water hypoxic, and continue to stimulate for 30 minutes ( Figure 5 -B), then the seawater was replaced, and the male gonads obtained by dissection were crushed using a 300-mesh sieve in clean seawater to obtain sperm for induction ( Figure 5 -D);

[0066] Shade drying + sperm induction: Place the sea cucumber in a basket and dry it in the shade for 30 minutes, then put it in a bucket filled with clean seawater, add sperm obtained by dissection to induce labor, and the amount of sperm added should be based on the water being slightly milky white;

[0067] Shade drying + temperature stimulation + sperm induction and induced labor: Place the sea cucumber in a basket and dry it in the shade for 30 minutes, transfer it to a bucket filled with clean seawater, slowly add hot fresh water, stir while adding, raise the water temperature from 25℃ to 30℃, and reduce the salinity from 31 to 29, add sperm obtained by dissection to induce induced labor, and the amount of sperm added is based on the water body being slightly milky white;

[0068] Temporary rearing and recovery of parent ginseng: After induced labor, parent ginseng was returned to the temporary rearing pond for a period of time. During this period, the breeding environment and food of parent ginseng were the same as above. After 3, 4 and 7 weeks of temporary rearing, shade drying for 30 minutes + sodium sulfite anoxic stimulation for 30 minutes + sperm induction were used to induce labor; after the operation of Example 4 of the present invention, compared with the traditional shade drying method, temperature stimulation method and sperm induction method, shade drying + sodium sulfite anoxic stimulation + sperm induction can observe spawning behavior within 2-4 hours of induced labor, and the number of eggs laid is more. In addition, in November, the water temperature dropped to 24-25°C and induced labor can still be successfully induced. A total of 5 induced labor experiments were carried out, and the success rate reached 100%, which far exceeded the ordinary shade drying and running water stimulation method. About 25 million fertilized eggs were obtained, and the gamete fertilization rate reached more than 95%. Most of the fertilized eggs developed normally. The parent ginseng gonads can be stored for a long time in the indoor pool. In December, the dissection found that the gonads of some female parent ginsengs were still full ( Figure 4 ).

[0069] Table 1 Comparison of the induced labor effects of sea cucumbers and ginseng

[0070]

[0071]

[0072] Note: The parents for ripening in indoor pools were caught by diving in the Judiaosha area of ​​Daya Bay on July 11; the parents of Daya Bay Catch 1 were caught by diving in the Judiaosha area of ​​Daya Bay on September 11; the parents of Dapeng Bay Catch 2 were caught by diving in the Dapeng Bay area of ​​Shenzhen on September 17; the parents of Dapeng Bay Catch 3 were caught by diving in the Dapeng Bay area of ​​Shenzhen on September 22; the parents for temporary rearing in indoor pools are the parents of Daya Bay Catch 2 and Dapeng Bay Catch 3, which are temporarily reared in greenhouse pools ( Figure 3 );DO<0.5mg L when using sodium sulfite hypoxia stimulation -1 ; - means not counted.

[0073] Example 5: Using an optimized method to induce spawning in brown sea cucumbers

[0074] Brown-ringed sea cucumber (Holothuria fuscocinerea) belongs to the genus Holothuria of the family Holothuridae. It is an important tropical sea cucumber. It is naturally distributed in the tropical Pacific region. It is distributed in China, Guam, the Philippines, Singapore, Indonesia and other places in the northwest Pacific region along the coast of the Gulf of Mexico, northern Australia, and the northwest Pacific region. The brown-ringed sea cucumbers in the South my country Sea are mainly distributed in the shallow sea reef areas of Taiwan, Guangdong, Hainan and the Xisha Islands. In the waters of Daya Bay in Shenzhen, the brown-ringed sea cucumber is a common species of sea cucumber. It often lives on the bottom of corals or reefs and feeds on debris and sediments. It is reported that artificial breeding of brown-ringed sea cucumbers has not yet been achieved, mainly because an effective method of inducing labor has not been developed. This species of sea cucumber is sensitive to environmental changes. The traditional shade drying method can easily damage the epidermis of the sea cucumber, and the induction effect is extremely poor. No successful cases of induction of labor have been reported. From June to July 2024, an artificial induction experiment of brown-ringed sea cucumbers was carried out at the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences:

[0075] Collection and temporary rearing of ginseng: On June 1, 2024, ginseng was caught in the Daya Bay waters and placed in indoor breeding ponds for temporary rearing. During this period, they were fed with sea mud and Sargassum powder, and long-flowing water was used to maintain stable water quality.

[0076] Induced labor of sea cucumbers: On June 24, 2024, the sea cucumbers were dried in the shade for half an hour + stimulated with sodium sulfite for half an hour + sperm induction for induction. The specific method was: the sea cucumbers were placed in a basket and dried in the shade for 30 minutes, 0.1g / L sodium sulfite was added to make the water hypoxic, and the stimulation continued for 30 minutes. After that, the sea water was replaced, and sperm obtained by dissection was used for induction. A total of 30 sea cucumbers were induced to labor, and 4 sea cucumbers produced sperm but no eggs. The epidermis of sea cucumbers is easily damaged after drying in the shade. When they were put back into the water, it was found that the sea cucumbers inhaled air and most of them floated on the water. Therefore, we speculate that the shade drying method is not conducive to the induction of labor of brown ring sea cucumbers. On June 25, 2024, sodium sulfite was used to stimulate for half an hour + sperm was used to induce induced labor (the parent sea cucumbers were transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to make the water hypoxic. The stimulation was continued for 30 minutes, and then clean seawater was replaced. Sperm obtained by dissection was added to induce induced labor. The amount of sperm added was based on the water being slightly milky white). There were a total of 25 sea cucumbers in the same batch of sea cucumbers, of which 5 sea cucumbers ejaculated and 4 ovulated. A total of 1.6 million fertilized eggs were obtained, with a fertilization rate of nearly 100%, and the larvae developed normally. After two weeks of temporary rearing, the induction of labor was continued. On July 9, 2024, sodium sulfite stimulation was used for half an hour + sperm induced induction of labor (the parent sea cucumber was transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to make the water body hypoxic. The stimulation lasted for 30 minutes, and then clean seawater was replaced, and sperm obtained by dissection was added to induce induction of labor). There were 20 sea cucumbers in the same batch, and 6 sea cucumbers ejaculated without ovulation. On July 10, 2024, sodium sulfite stimulation was used for half an hour + sperm induced induction of labor (the parent sea cucumber was transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to make the water body hypoxic. The stimulation lasted for 30 minutes, and then clean seawater was replaced, and sperm obtained by dissection was added to induce induction of labor). There were 18 sea cucumbers in the same batch, and 3 sea cucumbers ejaculated without ovulation. This method has a certain effect on inducing labor in brown-ringed sea cucumbers. When dissecting sea cucumbers, it was found that the color of the female gonads gradually faded. It is speculated that the gonads of brown-ringed sea cucumbers cannot be preserved for a long time in indoor environments. Therefore, based on the characteristics of brown-ringed sea cucumbers (easily damaged epidermis), the induction method of jade-footed sea cucumbers was improved (only sodium sulfite hypoxia stimulation + sperm induction was retained), which can induce labor in this difficult-to-induce sea cucumber, achieving a "0" breakthrough.

[0077] Example 6 Highly Efficient Artificial Cultivation of Sea Cucumber Larvae

[0078] From September to November 2023, an artificial breeding experiment of sea cucumbers will be conducted at the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The relevant process is as follows:

[0079] Water quality control: The natural seawater used for seedling cultivation is taken from the sea area near the experimental station. After testing, the content of various pollutants is lower than the second-class seawater quality standard. The extracted seawater is filtered through sand and then filtered with a filter bag with a pore size of 5 microns and treated with ultraviolet sterilization. During this experiment, the salinity range of the treated seawater was 28-33, and the temperature range was 28-31℃.

[0080] Induced spawning of Yuzu sea cucumber: The Yuzu sea cucumber was caught from the Dapeng Bay waters of Shenzhen, but due to the influence of the typhoon, anatomical observations found that 50% of the gonads of the parents had degenerated, and the gonads of the remaining individuals were very full. After 4 days of temporary rearing, induced spawning on September 21, 2023: First, the sea cucumber was removed from the temporary rearing pond and dried in the shade for 30 minutes, and then transferred to a plastic bucket filled with filtered seawater. 0.1g / L sodium sulfite was added to make the water body hypoxic. The stimulation continued for 30 minutes, and then the seawater was replaced. The sperm obtained by the dissection method was used for induction. The amount of sperm added was based on the water body being slightly milky white. After 3 hours, the sea cucumber was observed to ejaculate a large amount of sperm, followed by ovulation. After the ejaculation and ovulation process is over, the fertilized eggs are collected. First, use a 100-mesh sieve to filter out large particles of impurities, and then use a 400-mesh sieve to collect the fertilized eggs ( Figure 6 ), and remove excess sperm at the same time. The fertilized eggs are then placed in several 500L barrels for incubation. The hatching density is 0.5-0.7 / mL. Appropriate light has no adverse effects on planktonic larvae, but direct sunlight should be avoided. Continuous aeration should be used to keep the fertilized eggs suspended.

[0081] Cultivation method of planktonic larvae of sea cucumber: On the second day, the planktonic larvae of sea cucumber develop into the stage of small-eared larvae. At this time, the larvae have opened their mouths to eat. During the cultivation of planktonic larvae, 1 / 2 of the water is changed every 5-7 days according to the turbidity of the water body. When changing the water, stop aerating. Most of the larvae will swim to the surface of the water body. Use the siphon method to absorb the water at the bottom of the bucket. During this period, shake the siphon up and down appropriately to reduce damage to the larvae;

[0082] Feeding starts as soon as small-eared larvae are observed. Before the larvae develop to the late middle-eared larvae stage, the feeding amount is 2L / 1000L of Chaetoceros algae and 2L / 3000L of baker's yeast suspension each time. Feed once in the early morning and afternoon. Observe the feeding situation of the larvae 3-4 hours after feeding and adjust the feeding amount appropriately. From large-eared larvae to bottle-shaped larvae, the feeding amount is increased to 3L / 1,000L of Chaetoceros algae and 1L / 1,000L of baker's yeast suspension. Then, as the larvae in the water body gradually metamorphose and attach, the feeding amount is gradually reduced. Finally, five-tentacle larvae ( Figure 7 , Figure 8 ). We have found through long-term practice that when the density of planktonic larvae in the water is high, placing attachment substrates will cause a large number of larvae to sink to the bottom and die ( Fig.10), so when most of the planktonic larvae have metamorphosed into bottle-shaped larvae, reduce the amount of inflation appropriately, continue to culture until the density of larvae in the water body decreases significantly, and then start to release polyethylene corrugated board attachment base, the number of which is 2-3 frames / m 2 , which can effectively reduce the death of juveniles. The corrugated board is disinfected and attached with diatoms before use. In order to improve the attachment effect of juveniles, diatom attachment must be carried out more than one week in advance. The side with a larger area of ​​the corrugated board is attached to the bottom of the barrel. Later, the juvenile ginseng at the bottom of the barrel will disperse to the attachment base by itself;

[0083] Feeding too much bait at one time will increase the impurities at the bottom of the barrel and the suspended particles in the water, inhibiting the development of the larvae. Therefore, the amount of bait fed each time should be appropriate, and it can be fed less and more times. The larvae are cultivated with Chaetoceros muelleri and baker's yeast as bait. The density of Chaetoceros used is not less than 2 million / mL. Before feeding, the bait should be checked under a microscope to see if it is contaminated by protozoa. Normal algae water is rich and bright in color, without obvious odor. Avoid feeding aging and deteriorated bait. When too many impurities are found at the bottom of the barrel, the bottom should be sucked in time to remove dead larvae, residual bait and feces.

[0084] Intermediate cultivation of sea cucumbers: After the corrugated board is deployed, the bait is converted into benthic diatoms, marine red yeast, chlorella powder, sargassum powder and sea mud ( Fig.11 The sea cucumber starter feed is a mixture of marine red yeast, chlorella powder and Sargassum powder in a ratio of 1:1:2. Use a 160-mesh sieve to crush the algae powder in clean seawater, stir evenly and feed once a day. The feeding amount increases from 1ppm to 5ppm as the seedlings grow. Feed once every 3 days with the flushing liquid of benthic diatoms ( Fig. 9 ), the amount of feed also increases with the growth of the seedlings. Two weeks after the sea cucumbers attach, sea mud is added. The sea mud used is the floating mud on the surface of the intertidal zone at low tide. Use a sieve to filter out large particles and evenly sprinkle it on the bottom of the culture barrel every day to cover it with a layer of bottom mud ( Fig.14 ), use long-flowing water to keep the water quality stable 3 hours after feeding, and increase the number of daily feedings during the intermediate cultivation period to increase the growth rate of sea cucumbers, and continue to cultivate large-sized sea cucumber seedlings ( Fig.12 ). At the same time, the growth rates of juvenile sea cucumbers with and without sea mud were compared. The benthic diatoms, algae powder and yeast fed to the seedlings of the group without sea mud were the same as those of the group with sea mud, but the growth rate was significantly slower ( Fig.13 );

[0085] On April 12, 2024, a feeding experiment was conducted on young sea cucumbers using sea mud and sea sand ( Fig.14 , Fig.15), sea mud + Sargassum powder, sea sand + Sargassum powder were used as bait for young sea cucumbers. Seven small sea cucumbers were placed in each bucket, with 3 replicates in each group. The culture was carried out for one month, during which the water was changed once a week, with 1 / 2 of the water changed each time. Except for the initial addition of bait, no more bait was added afterwards. After the experiment, the weight and survival rate of sea cucumbers were measured. It was found that the growth effect of young sea cucumbers in the sea mud + Sargassum powder and sea sand + Sargassum powder groups was better, while the effect of using sea mud or sea sand alone was not good (Table 2).

[0086] Table 2

[0087]

[0088] Note: For the same row, different superscript letters indicate significant differences among the data groups (P<0.05).

[0089] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for inducing labor of sea cucumber, characterized in that: The following steps are involved: The sea cucumbers with mature gonads are first dried in the shade and then transferred to seawater. Sodium sulfite is added to make the water hypoxic to stimulate induced labor.

2. The method for inducing labor according to claim 1, characterized in that: After adding sodium sulfite to create oxygen deficiency in the water and stimulate induced labor, the seawater is replaced and sperm from sea cucumbers is added for induction.

3. The method for inducing labor according to claim 1, characterized in that: The drying time is 20-30 minutes.

4. The method for inducing labor according to claim 3, characterized in that: The drying in the shade takes 30 minutes.

5. The method for inducing labor according to claim 1, characterized in that: Add 0.1g / L sodium sulfite to make the water hypoxic and stimulate labor for 20-30 minutes.

6. The method for inducing labor according to claim 5, characterized in that: Stimulate labor for 30 minutes.

7. A method for efficient artificial breeding and intermediate cultivation, characterized in that: Including water quality control, ginseng ripening and spawning, pest control, planktonic larvae cultivation and attachment, and juvenile ginseng intermediate culture; The described induced labor by ginseng is the induced labor method of the above-mentioned sea cucumber.

8. The method according to claim 7, characterized in that The specific steps include: A. Water quality control: The natural seawater used must meet the second-class seawater quality standard and must be filtered and sterilized by ultraviolet light before use; B. Ripening and induced spawning of sea cucumbers: Wild sea cucumbers are collected in advance before the breeding season as a breeding group, and the parent sea cucumbers are temporarily kept in a ripening pool in a greenhouse. The water temperature in the ripening pool is higher than that in the natural sea area. The sea cucumbers feed on benthic diatoms in the pool. At the same time, they are fed with sea mud and Sargassum powder as supplementary bait to promote the maturation of the sea cucumber gonads. The method of inducing spawning is to place the parent sea cucumbers in a basket to dry in the shade for 30 minutes, and then transfer them to a plastic bucket filled with filtered seawater, add sodium sulfite to make the water body hypoxic, and continue to stimulate for 20-30 minutes. After that, the seawater is replaced, and the sperm obtained by dissection is used for induction. The parent sea cucumbers will lay eggs after 2-4 hours, and the parent sea cucumbers will be removed after they stop ovulating. The fertilized eggs are filtered through a 100-mesh silk screen to remove large impurities, and then concentrated through a 400-mesh silk screen to remove most of the excess sperm, and the fertilized egg density is adjusted to 0.5-0.7 eggs / mL for hatching and larval cultivation; C. Cultivation of juvenile sea cucumbers: The juvenile sea cucumbers are cultivated in a closed environment indoors. During the entire cultivation process, moderate aeration is maintained to keep the juveniles in a suspended state. They are fed with a certain ratio of Chaetoceros liquid and baker's yeast suspension. According to the turbidity of the aquaculture water, 1 / 2 of the water is replaced by siphoning every 5-7 days. Dead juveniles, residual bait and feces at the bottom of the bucket are regularly removed by siphoning. D. Sea cucumber larvae attachment and intermediate growth: When most of the larvae develop into barrel-shaped larvae, reduce the amount of inflation appropriately, and continue to culture until the number of planktonic larvae in the water body decreases significantly, then start to release polyethylene corrugated board attachment base, with a density of 2-3 frames / m 2 ; After the sea cucumber larvae attach, the feed is changed to benthic diatoms, marine red yeast, Chlorella powder, and Sargassum powder. Marine red yeast, Chlorella powder, and Sargassum powder are mixed in a mass ratio of 1:1:2 and fed once a day. The feed amount increases from 1ppm to 5ppm as the seedlings grow. Benthic diatoms are fed once every 3 days, and the feed amount also increases with the growth of the seedlings. After 3 hours of feeding, long-flowing water is used to keep the water quality stable. Two weeks after attachment, when the body length of the juvenile sea cucumbers reaches about 1mm, an appropriate amount of fresh sea mud is added daily. During the intermediate cultivation period, the number of daily feedings can be increased to increase the growth rate of the sea cucumbers, and large-sized sea cucumbers can be continuously cultivated.

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